GSM6456058: HCT116 ChIPseq Dyskerin control rep2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
DKC1
Cell type
Cell type Class
Digestive tract
Cell type
HCT 116
Primary Tissue
Colon
Tissue Diagnosis
Carcinoma
Attributes by original data submitter
Sample
source_name
HCT116
cell line
HCT116
cell type
Human colorectal carcinoma
treatment
Vehicle
chip antibody
dyskerin (Abnova, H00001736-m04)
geo_loc_name
missing
collection_date
missing
Sequenced DNA Library
library_name
GSM6456058
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
6 to 10 million cells were crosslinked for 5 minutes at room temperature with 16% methanol-free formaldehyde (Thermo Fisher Scientific) used at a final concentration of 1%. After quenching the reaction with 125 mM glycine final concentration, cells were washed twice in ice-cold PBS and collected by scraping. Pellets were resuspended in buffer A (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40 substitute) and incubated on ice for 10 minutes. After centrifugation (5000 rpm for 5 minutes at 4°C), the supernatants containing the cytoplasm were removed and the nuclei-containing pellets were washed in buffer A without detergent. Pellets were then resuspended in shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.1% SDS) and sonicated with a ME220 focused-ultrasonicator (Covaris). After clarification by centrifugation (13000 rpm for 10 minutes at 4°C), the supernatant was adjusted in composition to RIPA buffer and transferred to a new tube to be used for immunoprecipitation. Equal amounts of chromatin (as measured by DNA concentration) were incubated rotating overnight at 4°C with pre-conjugated beads (20 μl of Dynabeads and 3 μg antibody, dyskerin (Abnova, H00001736-m04) or RNAPII (MBL, MAB0601)) in RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 200 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS). Immunoprecipitates were washed once with each of the following: RIPA buffer, RIPA buffer high salt (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LCiCl, 0.5% NP-40 substitute, 0.5% sodium deoxycholate), and twice with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The beads were then resuspended in TE buffer with 0.5% SDS supplemented with proteinase K (New England Biolabs) and RNase A (Thermo Fisher Scientific) and incubated with shaking at 65°C for 4 hours. The DNA was recovered by phenol:chloroform:isoamyl alcohol extraction followed by ethanol precipitation using standard procedures, and resuspended in TE buffer. Libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer's instructions.